Synthesis and plant growth promoting activity of polyhydroxylated ketones bearing the 5α-hydroxy-6-oxo moiety and cholestane side chain

Article abstract of DOI:10.1016/j.steroids.2012.01.004

Three polyhydroxylated ketones bearing the 5α-hydroxy-6-oxo moiety were obtained from cholesterol. Two of them show plant growth promoting activity in the bean's second internode bioassay. The obtained results indicate that the presence of the 5α-hydroxy-

Full text of DOI:10.1016/j.steroids.2012.01.004

Steroids 77 (2012) 461–466
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and plant growth promoting activity of polyhydroxylated ketones
bearing the 5a-hydroxy-6-oxo moiety and cholestane side chain
Anielka Rosado-Abón a, Guadalupe de Dios-Bravo b, Rogelio Rodríguez-Sotres a,
Martín A. Iglesias-Arteaga a,
⇑
a Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF., Mexico
b Universidad Autónoma de la Ciudad de México Plantel San Lorenzo Tezonco Prol. Sn. Isidro 151 Col. Sn. Lorenzo Tezonco CP. 09790 México, DF., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Three polyhydroxylated ketones bearing the 5a-hydroxy-6-oxo moiety were obtained from cholesterol.
Received 9 December 2011
Received in revised form 3 January 2012
Accepted 5 January 2012
Two of them show plant growth promoting activity in the bean’s second internode bioassay. The obtained
results indicate that the presence of the 5 -hydroxy-6-oxo moiety may be capable to induce plant
growth promotion even the absence oxygenated functions in the side chain.
Ó 2012 Elsevier Inc. All rights reserved.
a
Available online 17 January 2012
Keywords:
Brassinosteroid analogs
Bean second internode bioassay
5a-Hydroxy-6-oxo steroids
Plant growth promoting activity
Cholesterol
1. Introduction
shown low to moderate, or even high plant growth stimulating
activity [14]. Interestingly our recently reported BA 10, which
Brassinolide (1) and other members of the family of brassi-
nosteroids (Bs) (2–6) (Fig. 1) are considered as the sixth class
of plant hormones [1–3]. In general, those compounds are char-
acterized by the presence of oxygenated functions in the rings A
and B as well as the side chain. The slight structural differences
that may be found between the naturally occurring Bs are
known to produce moderate to drastic variations in the plant
growth stimulating activity. This has led to the establishment
of generally accepted structural requirements for a good plant
growth stimulating activity [1].
Nevertheless, the fact that a wide variety of synthetic com-
pounds bearing minor, moderate or even drastic structural modifi-
cations have shown low to moderate, and in some cases, high plant
growth stimulating activity [2–15] suggests that the requirements
for a good biological activity are more flexible and variable than
those initially established [1]. Several books and reviews have col-
lected and analyzed the profuse available data and account for the
former statement [2–5,8] (See for review, [5]).
bears the 5a-hydroxy-6-oxo moiety and a furostane side chain also
showed moderate biological activity [15] (Fig. 2).
After more than 30 years of brassinosteroids research, two main
tasks have become evident:
(a) The synthesis of naturally occurring brassinosteroids and highly
active analogs. Since this activity is directed to studies on
structure–activity, modes of action (interaction substrate–
receptor, plant physiology, genetics, etc.) the compounds
conceived for this task are expected to bear no – or only
minor structural modifications on the known bioactive
functions.
(b) The search for promising candidates for the massive applica-
tion in agriculture. In this context and considering that sur-
prises occur, this activity needs a much more open search
that should include simple (and even known) compounds
with structural modifications that simplify the synthetic
procedure and whose plant growth stimulating activity
have been not studied. This does not mean a comeback to
the trial and error system, but an open search that com-
bines the cumulated experience and some audacity. In
addition, the results would feedback task A that may try
to explain why some given compounds with drastically
modified structures display a significant plant growth stim-
ulating activity.
In particular Brosa and coworkers as well as Galagowsky have
described the synthesis of several brassinosteroids analogs (BA)
(i.e. 7–9, Fig. 2) bearing a 5a-hydroxy-6-oxo moiety that have
⇑
Corresponding author. Tel./fax: +52 55 56223803.
E-mail address: martin.iglesias@unam.mx (M.A. Iglesias-Arteaga).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.01.004

462
A. Rosado-Abón et al. / Steroids 77 (2012) 461–466
HO
HO
HO
OH
OH
HO
OH
HO
HO
HO
HO
HO
O
H
O
H
O
O
H
O
O
dolicholide (3)
brassinolide (1)
homobrassinolide (2)
OH
OH
OH
OH
OH
OH
HO
HO
HO
HO
H
O
H
H
O
O
castasterone (4)
typhasterol (5)
teasterone (6)
Fig. 1. Some naturally occurring brassinosteroids.
OH
OH
OH
OH
HO
HO
HO
HO
OH
OH
O
O
7
8
OH
OH
HO
O
HO
HO
HO
HO
OH
O
O
9
10
Fig. 2. Some BA bearing the 5a-hydroxy-6-oxo moiety.
whose prohibitive prices preclude the massive and generalized
application of brassinosteroids to agriculture.
Those facts have prompted us to initiate a program for the prep-
aration and evaluation of the plant growth promoting activity of a
series of simple compounds with the easy to obtain 5a-hydroxy-6-
oxo moiety and different side chains, using cheap and readily avail-
Since the introduction of the 5a-hydroxy-6-oxo moiety starting
from 3b-hydroxy-D5 steroids is much easier than the preparation
of the B-homo-7-oxa-6-oxo moiety that characterizes the more ac-
tive compounds, BA bearing the 5
in general, more suitable for large scale preparation. Hence, agri-
cultural applications of moderately bioactive BA bearing the 5
a-hydroxy-6-oxo moiety may be,
a-
able steroids as starting materials. Herein we report on an opti-
mized synthesis of three 5a-hydroxy-6-oxo-steroids derived from
hydroxy-6-oxo moiety may, at the end, prevail over that of the
highly active commercially available B-homo-7-oxa-6-oxo analogs

A. Rosado-Abón et al. / Steroids 77 (2012) 461–466
463
cholesterol and the study of their plant growth stimulating activity
in the bean second internode bioassay.
66.8 C-3, 35.5 C-4, 80.0 C-5, 214.5 C-6, 41.7 C-7, 37.4 C-8, 44.2 C-
9, 42.4 C-10, 21.3 C-11, 39.4 C-12, 43.0 C-13, 56.2 C-14, 23.8 C-
15, 29.7 C-16, 56.0 C-17, 11.9 C-18, 13.8 C-19, 35.6 C-20, 18.5 C-
21, 36.0 C-22, 23.7 C-23, 39.5 C-24, 27.9 C-25, 22.4 C-26, 22.7 C-
27. MS (EI, 70 eV): 420 MHþ2 , 419 MH+, 418 M+, 401, 400, 385,
367, 318 (100%), 303.
2. Experimental
2.1. General conditions
Reactions were monitored by TLC on ALUGRAMÒ SIL G/UV254
plates from MACHEREY–NAGEL. Chromatographic plates were
sprayed with a 1% solution of vanillin in 50% HClO4 and heated un-
til color developed. Melting points were measured on a Melt-Temp
II equipment and are uncorrected. Mass spectra were registered in
a Thermo-Electron spectrometer model DFS (Double Focus Sector).
NMR spectra were recorded in CDCl3 or DMSO-d6 solution in Var-
ian INOVA 400 or 300 MHz spectrometers using the solvent signals
as references. NMR signals assignments were made with the aid of
DEPT and a combination of 2D homonuclear (1H–1H) and hetero-
nuclear (1H–13C) correlation techniques, which included 1H–1H
COSY, 1H–1H Nuclear Overhauser Effect Spectroscopy (NOESY),
and Heteronuclear Single Quantum Correlation (HSQC). All 2D
NMR spectra were recorded using the standard pulse sequences
and parameters recommended by the manufacturer.
2.2.3. 5-Hydroxy-5a-cholest-2-en-6-one (14)
Tosyl chloride (953.2 mg, 5 mmol) was added to a solution of
the dihydroxylated ketone 13 (1 g, 2.39 mmol) in dry pyridine
(12 ml), the mixture was stirred overnight at room temperature
and poured into 3% HCl/ice, the solid product was filtered off,
washed with water, dissolved in ethyl acetate, dried (Na2SO4)
and evaporated. The dried solid was refluxed for 30 min. in dry
DMF (25 ml) with LiBr (1.3195 g) and Li2CO3 (1.0925 g) and the
mixture was allowed to cold to room temperature. Ethyl acetate
(70 ml) was added, the mixture was filtered, the solid residue
was washed with ethyl acetate (4ꢀ 5 ml) and the organic layer
was washed with water (6ꢀ 20 ml), dried (Na2SO4) and evapo-
rated. The residue was purified in a chromatographic column (hex-
ane/ethyl acetate 8/1) to afford the unsaturated 5a-hydroxyketone
14 (0.7315 g, 1.83 mmol, 77%). M.p 134–136 °C (from ethyl acetate);
Lit. 140–141.5 °C [17]. 1H NMR (300 MHz, CDCl3), d ppm: 5.69–
5.57 (2H, m, H-2, H-3), 2.68 (1H, dd, J = 12.6 Hz, 12.6 Hz, H-7ax),
2.18 (1H, dd, J = 4.1 Hz, 12.7 Hz, H-7eq), 0.90 (3H, d, J = 6.5 Hz,
CH3-21), 0.85 (6H, dd, J = 1.4 Hz, 6.6 Hz, CH3-26 and CH3-27),
0.69 (3H, s, CH3-19), 0.64 (3H, s, CH3-18). 13C NMR (75.5 MHz,
CDCl3), d ppm: 28.0 C-1, 122.4 C-2, 125.5 C-3, 34.5 C-4, 78.0 C-5,
211.5 C-6, 42.6 C-7, 37.5 C-8, 45.2 C-9, 42.3 C-10, 21.0 C-11, 39.5
C-12, 42.9 C-13, 56.4 C-14, 23.8 C-15, 30.1 C-16, 56.1 C-17, 11.9
C-18, 14.5 C-19, 35.7 C-20, 18.6 C-21, 36.1 C-22, 23.9 C-23, 39.6
C-24, 28.0 C-25, 22.5 C-26, 22.8 C-27. MS (EI, 70 eV): 402 MHþ2 ,
MH+, 400 M+, 385, 368, 367 (100%), 355.
2.2. Synthetic procedures
2.2.1. 3b-Acetoxy-5-hydroxy-5a-cholestan-6-one (12)
m-CPBA (0.968 g, 5.61 mmol) was added to a solution of cho-
lesteryl acetate (11) (1.7148 g, 4 mmol) in CH2Cl2 (20 ml) and the
mixture was stirred until the starting material disappeared (1–
1.5 h, TLC). Acetone (50 ml) was added and the mixture was cooled
to 0 °C in an ice bath before addition of a solution of CrO3 (1.4286 g,
14.29 mmol) in water (4.3 ml) The ice bath was removed, the mix-
ture was stirred at room temperature for 20 min and cooled to 0 °C
in the ice bath prior to drop wise addition of a solution of CrO3
(0.7143 g, 7.14 mmol) in water (2.15 ml). The ice bath was re-
moved and the mixture stirred for 50 min, before addition of water
(50 ml) and extraction with ethyl acetate (2ꢀ 50 ml). The organic
layer was washed with water (9ꢀ 50 ml), 10% NaHCO3 solution
(5ꢀ 50 ml), water (2ꢀ 50 ml) and saturated NaCl solution
(1ꢀ 50 ml), dried (Na2SO4) and evaporated to afford the desired
ketol 12 (1.7597 g, 3.82 mmol, 96%). Mp 230–232 °C (from ethyl
acetate); Lit. 232–233 °C [16]. 1H NMR (300 MHz, CDCl3), d ppm:
5.05–4.94 (m, 1H, H-3), 2.78 (dd, J = 12.6, 12.6 Hz, 1H, H-7 ax.),
2.01 (s, 3H, CH3COO-3), 0.91 (d, J = 6.3 Hz, 3H, H-21), 0.87 and
0.85 (d, J = 6.6 Hz, 3H each, H-26 and H-27), 0.81 (s, 3H, H-19),
0.64 (s, 3H, H-18). 13C NMR (75.5 MHz), d ppm: 29.6 C-1, 26.4 C-
2, 71.5 C-3, 32.0 C-4, 79.8 C-5, 213.8 C-6, 41.8 C-7, 37.5 C-8, 44.3
C-9, 42.6 C-10, 21.5 C-11, 39.7 C-12, 43.2 C-13, 56.4 C-14, 24.0 C-
15, 28.2 C-16, 56.2 C-17, 12.1 C-18, 13.9 C-19, 35.8 C-20, 18.7 C-
2.2.4. 2
a,3
a,5-Trihydroxy-5a-cholestan-6-one (15) and 2b,3b,5-
trihydroxy-5a-cholestan-6-one (16)
N-methylmorpholine N-oxide (0.243 g) and a 12.5 mg/ml solu-
tion of OsO4 in tert-butyl alcohol (0.39 ml) were added to a solution
of the unsaturated ketol 14 (200 mg, 0.5 mmol) in THF (5 ml) and
the mixture was stirred overnight under N2 atmosphere before
addition of a solution of Na2SO3 (0.4 g) in water (3 ml). The mixture
was extracted with ethyl acetate (2ꢀ 50 ml) and the organic layer
was washed with brine (5ꢀ 20 ml), dried and evaporated to afford
a mixture of the trihydroxylated compounds 15 and 16 that were
separated in a chromatographic column using 1/1 of hexane/ethyl
acetate as eluent.
2.2.4.1. 2a,3a,5-Trihydroxy-5a-cholestan-6-one (15). Yield (134.4
21, 36.2 C-22, 24.0 C-23, 39.6 C-24, 28.1 C-25, 22.6 C-26, 22.9 C-
mg, 0.31 mmol, 62%). M.p 190–192 °C (from ethyl acetate) [18].
1H NMR (400 MHz, DMSO-d6), d ppm: 5.61 (1H, s, OH-5), 5.44
(1H, d, J = 3.0 Hz, OH-3), 4.54 (1H, d, J = 5.8 Hz, OH-2), 3.92 (1H,
m, H-3), 3.63–3.59 (1H, m, H-2), 2.56 (1H, dd, J = 12.4 Hz,
12.4 Hz, H-7ax), 1.84 (1H, dd, J = 4.1 Hz, 12.4 Hz, H-7eq), 0.89 (3H,
d, J = 6.4 Hz, CH3-21), 0.84 (6H, dd, J = 1.9 Hz, 6.6 Hz, CH3-26 and
CH3-27), 0.67 (3H, s, CH3-19), 0.61 (3H, s, CH3-18). 13C NMR
(100 MHz, DMSO-d6), d ppm: 27.6 C-1, 66.4 C-2, 69.2 C-3, 34.3 C-
4, 79.1 C-5, 210.4 C-6, 40.9 C-7, 36.4 C-8, 44.1 C-9, 42.6 C-10,
20.6 C-11, 39.2 C-12, 44.6 C-13, 55.8 C-14, 23.4 C-15, 30.5 C-16,
55.4 C-17, 11.7 C-18, 14.3 C-19, 35.1 C-20, 18.4 C-21, 35.5 C-22,
23.2 C-23, 38.8þ C-24, 27.3 C-25, 22.3 C-26, 22.6 C-27. MS (IE,
70 eV): 436 MH2 , 435 MH+, 434 M+, 416, 398, 383, 367, 355, 331,
318, 317, 285, 247, 243, 231, 211, 189, 175, 161, 149, 137, 135,
109, 95, 81,69 (100%), 55. HRMS-EI: observed 434.3385; required
for C27H46O4 434.3391.
+
27, 171.8 CH3COO-3, 21.5 CH3COO-3. MS (EI, 70 eV): 460 M
418, 401, 400 (100%), 318.
,
2.2.2. 3b,5-Dihydroxy-5a-cholestan-6-one (13)
A saturated solution of K2CO3 in methanol (100 ml) was added
to the solid ketol 12 (2 g, 4.34 mmol) and the mixture was stirred
overnight at room temperature. Half the solvent was evaporated,
the mixture was poured into ice-water, the produced solid was fil-
tered off, washed with water and dried in the air to afford the dihy-
droxylated ketone 13 (1.3411 g, 3.21 mmol, 74%). M.p 230–232 °C
(from methanol); Lit. 231–232° [16]. 1H NMR (300 MHz, CDCl3), d
ppm: 4.62 (broad, H-4), 3.94–3.90 (m, H-3), 3.12 (broad, H-7eq),
2.78 (dd, J = 12.6 Hz, 12.6 Hz, H-7ax), 0.91 (d, J = 6.4 Hz, CH3-21),
0.86 (d, J = 6.4 Hz, CH3-26 and CH3-27), 0.77 (s, CH3-19), 0.64 (s,
CH3-18). 13C NMR (75.5 MHz, CDCl3), d ppm: 29.9 C-1, 28.0 C-2,

464
A. Rosado-Abón et al. / Steroids 77 (2012) 461–466
i
ii
HO
AcO
AcO
12
11
13
OH
OH
O
O
iii, iv
HO
HO
HO
v
+
HO
OH
OH
16
15
OH
14
O
O
O
i) a MCPBA/ CH2Cl2, b CrO3/H2O; ii) K2CO3/CH3OH;
iii) TsCl/pyr; iv) LiBr/ Li2CO3/ DMF reflux; v OsO4/NMMO/ THF
Scheme 1. Synthetic procedure.
H
CH3
OH
CH3
HO
H
H
HO
15
16
O
O
OH
OH
H
OH
Fig. 3. Selected NOE correlations in triols 15 and 16.
d, J = 6.2 Hz, OH-3), 4.08 (1H, d, J = 5.8 Hz, OH-2), 3.75 (2H, m, H-
2 and H-3), 2.66 (1H, dd, J = 12.5 Hz, 12.5 Hz, H-7ax), 1.82 (1H,
dd, J = 5.0 Hz, 12.7 Hz, H-7eq), 0.88 (3H, d, J = 6.5 Hz, CH3-21),
0.87 (3H, s, CH3-19), 0.84 (6H, dd, J = 2.0 Hz, 6.6 Hz, CH3-26 and
CH3-27), 0.60 (3H, s, CH3-18). 13C NMR (100 MHz, DMSO-d6), d
ppm: 27.7 C-1, 68.3 C-2, 67.1 C-3, 36.6 C-4, 79.4 C-5, 212.3 C-6,
41.2 C-7, 36.0 C-8, 44.1 C-9, 42.7 C-10, 21.0 C-11, 39.4 C-12, 41.5
C-13, 55.9 C-14, 23.5 C-15, 30.5 C-16, 55.6 C-17, 11.8 C-18, 15.7
C-19, 35.2 C-20, 18.5 C-21, 35.6 C-22, 23.2 C-23, 38.9þ C-24, 27.4
C-25, 22.4 C-26, 22.7 C-27. MS (EI, 70 eV): 436 MH2 , 435 MH+,
434 M+, 416, 390, 373, 355, 331, 318, 316, 303, 255, 247, 221,
175, 161, 147, 121, 137, 107, 95, 81, 69, 57 (100%). HRMS (EI,
70 eV): observed 434.3368, required for C27H46O4 434.3391.
2.3. Biological assay
2.3.1. Bean’s second internode elongation assay
The details of this test have been described elsewhere [15].
Briefly, bean seed were germinated in sterile wet paper towels. Bean
seedling were planted in small pots with agrolite and watered with
Hoghland solution for 2 weeks. Compounds were dissolved in a mix-
Fig. 4. Biological activity of the studied compounds. Homobrassinolide 2 (j), or
compounds 13 (d), 15 (N), or 16 (.). The abscissa scale is logarithmic, but zero
dosage was arbitrarily placed in the beginning of the graph. Length values are
plotted as differences from control plants treated with vehicle (lanolin), therefore at
zero dose, the length becomes zero.
ture of lanolin and ethanol, and 2 ll of the solution containing the
dose indicated was applied to the base of the second internode. After
48 h, the length of the second internode was measured. Groups of
10–15 plants were used for each treatment. The data are from three
independent experiments. Data is presented as length differences,
i.e. the second internode length was recorded and the median value
2.2.4.2. 2b,3b,5-Trihydroxy-5a-cholestan-6-one (16). Yield (62.3 mg,
0.14 mmol, 28%). M.p. 262–264 °C (from diethyl ether) [18]. 1H
NMR (400 MHz, DMSO-d6), d ppm: 5.33 (1H, s, OH-5), 4.29 (1H,

A. Rosado-Abón et al. / Steroids 77 (2012) 461–466
465
of the internode length for control plants was subtracted to all data.
For each compound tested, the overall statistical significance of the
differences between the treatments (different dosages) was tested
using the distribution-independent test of Kruskal–Wallis one way
analysis of variance (ANOVA on ranks), individual differences
against control were isolated with Dunn’s method.
Compounds 13 and 15 gave the maximal response (i.e. the high-
est internode length difference recorded for each compound)
nearly as good as the natural phytohormone (2), and their effect
at the lower dose tested (1 pg/plant) was at least as large as the ef-
fect observed with 2 (there was no statistically significant differ-
ence between the data for 2, and the data for the compounds 13
and 15 at this dose). Therefore, we can safely conclude that 13
and 15 were as effective as the natural phytohormone (2) in the
dosage range tested (Fig. 4 and Table 1).
3. Results and discussion
3.1. Chemistry
4. Conclusions
Cholesteryl acetate (11) was converted into the acetylated ketol
(12) following our recently published one-pot epoxidation-oxida-
tive cleavage procedure [19]. Saponification of 12 afforded the
dihydroxylated ketone 13 that on consecutive tosylation and elim-
ination afforded the unsaturated ketol 14. Dihydroxylation of 14
employing OsO4 and NMMO as co-oxidant produced a mixture of
Three brassinosteroid analogs bearing the 5a-hydroxy-6-oxo
moiety and different hydroxylated A-rings were obtained from
cholesterol. Compounds 13 and 15 showed interesting properties
as plant growth stimulators in the bean’s second internode elonga-
tion assay. The obtained results indicate that the presence of the
5a-hydroxy-6-oxo moiety, even in the absence of oxygenated
the 2a,3a and 2a,3b diols 15 and 16 (Scheme 1).
functions in the side chain, is capable to induce plant growth stim-
ulation. The active compounds 13 and 15 have the advantage that
can be easily obtained from the inexpensive and ready available
cholesterol acetate (11).
Although an analogous preparation of compounds 15 and 16
has been previously reported by Rodriguez and coworkers in the
synthesis of cytotoxic 6E-hydroximino-4-ene steroids [18], we
decided to carry out a complete high field NMR characterization
and provide unambiguous evidences on the orientation of the diol
moiety introduced at positions C-2 and C-3, not provided in the
above cited report. In particular, the orientation of the introduced
cis-diol moiety can be easily established in NOESY experiments.
Acknowledgements
We thank Dirección General de Asuntos del Personal Académico
(DGAPA-UNAM) for financial support via project IN221911 and
CONACyT (Mexico) for the scholarship granted to A.R-A. We are
also grateful to Instituto de Ciencia y Tecnología del Distrito Fed-
eral (ICyT DF) for the financial support. We are indebted to Profes-
sor Vladimir Khripach of the Academy of Sciences of Byelorussia
for the kind gift of homobrassinolide. Thanks are due to Nuria Estu-
rau-Escofet for registering all NMR spectra and to Georgina Duarte
Lisci and Margarita Guzmán Villanueva (USAI-UNAM) for register-
ing Mass spectra.
While in the 2
OHM5 -OH NOE effects indicate the
the 2bꢁoH MH-19 and H-3 M5 -oH NOE effects observed in the
2b,3b,5 -triol 16 evidence the b-orientation of the diol moiety at
a,3a,5
a-triol 15 the observed H-2bMH-19 and 3
a-
a
a-orientation of the 2,3-diol,
a
a
a
positions C-2 and C-3 (Fig. 3).
3.2. Biological activity
A number of recent findings on the Bs signal transduction path-
ways [20] have paved the way for more direct molecular tests of
the Bs effects. However, it has also been shown that these molec-
ular responses are not always proportional to the applied dose
[21]. Therefore, until a molecular test is properly validated, the
classic physiological assays continue to be more reliable. Hence,
the biological activities of compounds 13, 15 and 16 were deter-
mined employing the bean’s second internode elongation assay
using homobrassinolide (2) as active control, and the vehicle (lan-
olin) as a control lacking activity.
The compounds were tested at concentrations from 1 pg to 1 ng
per plant, using the bean second internode assay. From the non-
parametric statistical test of the data, homobrassinolide (2) and
compounds 13 and 15 were found to stimulate plant growth in
the employed assay. Compound 16 appeared to give some effect,
but the data lacked statistical significance, therefore, we consider
this compound as inactive.
References
[1] Cluter HG, Yokota T, Adam G, editors. Brassinosteroids. Chemistry bioactivity
and applications ACS Symp 474. Washington DC: American Chemical Society;
1991.
[2] Khripach VA, Zhabinskii VN, de Groot AE. Brassinosteroids A New Class of Plant
Hormones. Academic Press; 1999.
[3] Sakurai A, Yokota T, Clouse SD, editors. Brassinosteroids. Steroidal Plant
Hormones. Tokyo: Springer; 1999.
[4] Hayat S,editor. Brassinosteroids: bioactivity and crop productivity. Kluwer
Academic Publishers; 2003.
[5] Adam G, Marquardt V. Brassinosteroids. Phytochemistry 1986;25:1787–800.
[6] Iglesias-Arteaga MA, Leliebre Lara V, Pérez-Martínez CS, Coll F. Synthesis of
spirobrassinosteroids analogues of 6-desoxocastasterone. Quim Nova
1997;20:361–4.
[7] Iglesias-Arteaga MA, Pérez Gil R, Leliebre Lara V, Pérez-Martínez CS, Coll F.
Synthesis and biological activity of (22r,25r)-5
Chem Res S 1996:504–5.
a-furostan-2a,3a,26-triol. J
[8] Iglesias-Arteaga MA, Pérez R, Coll F. Natural brassinosteroids and synthetic
analogues. Cienc Quím 1996;27:3–12 (Chem. Abstr. 1997, 127, 248270).
[9] Iglesias-Arteaga MA, Pérez Gil R, Pérez-Martínez CS, Coll F. Spirostanic
analogues of teasterone. synthesis, characterization and biological activity of
laxogenin (23s)-hydroxylaxogenin and 23-ketolaxogenin (23-oxolaxogenin). J
Chem Soc Perkin Trans 1 2001;3:261–6.
[10] Iglesias-Arteaga MA, Pérez-Martínez CS, Coll F. Spirostanic analogues of
castasterone. Steroids 2002;67:159–63.
[11] Pérez Gil R, Iglesias-Arteaga MA, Pérez Martínez CS, Coll García D, Coll F.
Synthesis of analogues of brassinosteroids with 24-hydroxilated side chain.
Eur J Org Chem 1998;11:2405–7.
Table 1
Bean’s second internode elongation differences (mm).
Dose
(ng/
2
13
15
16
homobrassinolide
plant)
ˇ
[12] Šíša M, Hnilicková J, Swaczynová J, Kohout L. Syntheses of new androstane
0.001
0.010
0.100
1.000
1.01 0.28
2.25 0.44 (⁄) 2.18 0.29 (⁄) 0.72 0.22
0.72 0.15
2.61 0.37 (⁄) 1.26 0.25
1.56 0.28 1.39 0.24
brassinosteroids with 17b-ester group - butyrates, heptafluorobutyrates, and
laurates. Steroids 2005;70:755–62.
[13] Strnad M, Kohout L. A simple brassinolide analogue 2
2.83 0.68 (⁄)
2.09 0.41 (⁄)
1.86 0.22 (⁄)
2.04 0.45 (⁄) 1.60 0.40
2.15 0.64
1.48 0.26
a,3a-dihydroxy-17b-(3-
methyl-butyryloxy)-7-oxa-B-homo-5 -androstan-6-one which induces bean´ s
a
second internode splitting. Plant Growth Regul 2003;40:39–47.
[14] Brosa C, Soca L, Terricabras E, Ferrer JC, Alsina A. New synthetic
(⁄) Statistically significant difference from control in a Kruskal–Wallis ANOVA,
followed by the Dunn’s test (p < 0.05). The length of the control plants internode
was subtracted from that of the plants treated with the studied compounds.
brassinosteroids: a 5
a-hydroxy-6-ketone analog with strong plant growth
promoting activity. Tetrahedron 1998;54:12337–48;

466
A. Rosado-Abón et al. / Steroids 77 (2012) 461–466
Ramírez JA, Teme-Centurión OM, Gros EG, Galagovsky LR. Steroids
2000;65:329–37.
[18] Deive N, Rodriguez J, Jimenez C. Synthesis of cytotoxic 6E -hydroximino-4-ene
steroids: structure/activity studies. J Med Chem 2001;44:2612–8.
[15] Romero-Avila M, de Dios-Bravo G, Mendez-Stivalet JM, Rodríguez-Sotres R,
Iglesias-Arteaga MA. Synthesis and biological activity of furostanic analogues
[19] Rosado-Abón A, Romero-Avila M, Iglesias-Arteaga MA. A rapid and simple one-
pot procedure for the synthesis of 3b-acetoxy-5a-hydroxy-6-oxo steroids.
of brassinosteroids bearing the
2007;72:955–9.
5
a
-hydroxy-6-oxo moiety. Steroids
Arkivoc 2010;X:110–5.
[20] Kim TW, Wang ZY. Brassinosteroid signal transduction from receptor kinases
to transcription factors. Ann Rev Plant Biol 2010;61:681–704.
[21] Symons GM, Reid JB. Brassinosteroids, de-etiolation and the re-emerging art of
plant hormone quantification. Plant Signal Behav 2008;3:868–70.
[16] Fieser LF, Rajagopalan S. Selective oxidation with N-bromosuccinimideII.
Cholestane-3 ,5 ,6b-triol. J Am Chem Soc 1949;71:3938–41.
a
a
[17] Hans R, Walker FE, Collins RW.
J Org Chem 1951;16:1753–60.
D4 -cholestene-6-one and related compounds.